INVESTIGATION OF MICROBIAL POLLUTION OF SOME VEGETABLE SAMPLES COLLECTED FROM KONYA CITY MARKETS IN TURKEY

, microbiological quality of reclaimed wastewater is very important for any reuse in order to assure health protection preventing environmental degradation and avoiding public nuisance. Epidemiological investigations on reuse of raw or minimally-treated wastewater for food crop irrigation provided evidence of infectious diseases transmission. A case study was undertaken to investigate entrobacteria as an indicator of microbiological pollution in vegetables sold at three public markets in Konya Province.

MATERIAL AND METHOD

1. Description of the Area and the Market Places The area under examination, i.e. Konya Province, is located on a large plateau that is known “The Great Konya Basin” in the central part of Anatolia. The Province is situated at 36051″-39029′ Northern latitude and 31o36′ -34o52′ Eastern longitude and the surface area of the Basin is nearly 5.4 million hectares that makes up nearly 7% of land surface of Turkey. The vegetable specimens, as experimental material were acquired of the three main markets in Konya Province: Beysehir, Doğanhisar and Ilgıntowns which are subprovinces of Konya Province, in 2002 (Figure 1) Figure 1. Description of the Konya Province and its subprovinces from which the vegetable specimens have been collected.

2. Collection and Preparation of the Experimental Specimens The following vegetable varieties were studied: green (fresh) onion, tomatoes, cabbage, parsley, watercress, radishes, carrots, cucumber skin, lettuce, green (fresh) pepper. A total 20 specimens were taken of each product in each market. All specimens of the vegetables were raw and fresh when they were brought to the laboratory for the work. Four different disinfection treatments were used for the vegetable specimens studied. Some kind of hydroclonazone tablets were used to disinfect the specimens. The tablets were composed of chloramines. The specimens were also immersed in two different solutions containing 0.32 colloidal silver and chlorine salts respectively. The specimens were also washed with detergent and scalded with hot water in the laboratory. Five replications were used for each treatment applied in the study. After obtaining the vegetable specimens from the markets, 20 gm of each were weighed out. For each of these aliquots, one of the following treatments was chosen at random: (a) washed with detergent and ordinary water, (b) treated with a commercial bactericide, (c) scalded at 70 0C for 60 seconds, (d) washed with running tap water, or (e) the unwashed vegetable was taken with no treatment. After the foregoing treatment mentioned above, each specimens was liquefied in a blender with 200 mL of sterile water. They were then filtered through five layers of sterile gauze and the filtrate was collected in glass containers. Subsamples of 10 mL were added to

mL sterile physiological saline (0.85 %) water. After vigorous shaking the following microbiological analyses were performed by the membrane filtration technique. The diluted filtrates were immediately streaked on selected culture media such as EMB, Mac Conkey, and/or agar containing lactose. These were incubated 37 0C for 24 hr. Typical coliform and the suspected enterobacteria colonies were identified by their colonial morphology and smears which were stained with Gram’s stain. Colonies suspected of being pathogenic were streaked on brilliant green agar, S/S agar and/or deoxicholated citrate agar. These were all incubated under the same conditions; and differential media such as the Kligler’s iron agar, Sim’s agar, and/or surraco medium were used to identify the genus. Besides, total bacterial counts were executed on Plate Count Agar (OXOID) by pour plate techniques with an incubation of 15 days at 18 0C [3, 5, 8, 10]. All platings were done in triplicate.

RESULTS AND DISCUSSION All the microbiological data, obtained during this study, were summarized in Table I, II and III. The values in the Table I and II represent the distribution patterns of the total coliforms and pathogenic microorganisms i.e. Proteus sp., Salmonella sp. and Shigella sp. respectively. The results obtained from the Tables can be summarized as follows: − The distribution of the total coliform bacteria on the vegetable specimens ranged from 24 percent to 100 percent (Table I). − The highest incidence of coliform contamination ( approximately 50 %) was found with parsely specimens among all the vegetable specimens studied and it was followed by watercress (48-49 %), lettuce (48-50 %), radishes (46-48 %), green onions (46-48 %), carrots (39-49 %), cabbage (37-43 %), cucumber skin (36-41 %), tomatoes (36-39 %) and green pepper specimens (33-39%) respectively. So that the lowest figures related to green pepper specimens (Table I). Table I. Percent of vegetables collected from Beysehir, Doğanhisar and Ilgın markets in Konya Province that gave positive responses for Coliform bacteria (%), (n=5) The treatments used No treatment Washed with water Treated by scalding Washed with a bacteriocide Washed with a detergent Vegetable varieties Number of specimens BŞ DH IL BŞ DH IL BŞ DH IL BŞ DH IL BŞ DH IL ------------------------------------------------------------------------%------------------------------------------------------------------------- Green (fresh) pepper 20 33 38 39 32 34 37 14 18 26 20 27 32 25 28 31 Lettuce 20 48 49 50 47 49 49 34 35 36 35 38 39 41 45 47 Cucumber (skin) 20 36 38 41 35 37 40 13 19 35 33 29 31 35 37 39 Carrots 20 39 45 49 38 42 45 19 23 23 32 37 34 36 39 41 Radishes 20 46 47 48 45 46 42 32 34 40 38 39 40 46 47 50 Watercress 20 48 49 50 47 49 50 33 35 36 43 44 48 49 50 48 Parsley 20 50 50 50 47 49 50 33 35 36 44 46 47 47 49 48 Cabbage 20 37 42 43 32 42 44 12 16 23 23 29 34 26 34 37 Tomatoes 20 36 37 39 35 37 38 17 19 28 35 36 38 38 39 40 Green onions 20 46 46 48 45 46 47 34 39 38 40 43 45 40 45 46 BS: Beysehir Market Place, DH: Doğanhisar Market Place, IL: IlgınMarket Place Table II. Percent of vegetables collected from Beysehir, Doğanhisar and Ilgın Markets in Konya Province that gave positive responses for pathogenic microorganisms (%), (n=5) The treatments used No treatment Washed with water Treated by scalding Washed with a bacteriocide Washed with a detergent Vegetable varieties Number of specimens BŞ DH IL BŞ DH IL BŞ DH IL BŞ DH IL BŞ DH IL ------------------------------------------------------------------------%------------------------------------------------------------------------- Green (fresh) pepper 20 0 1s 2 0 0 1p 0 2s 2sh 0 1p 3 0 1s 2p Lettuce 20 6p 4s 3sh 7p 5s 9p 3p 5s 6sh 6p 5s 7sh 2 5s 6sh Cucumber (skin) 20 0 1s 2p 0 0 0 0 0 0 0 0 1p 0 3p 4p Carrots 20 0 0 1p 0 2p 2p 0 2p 4p 0 3p 2p 0 2p 2p Radishes 20 0 2p 1p 9p 2s 1p 0 2p 2p 0 2p 3p 0 0 0 Watercress 20 8p 5s 3p 9p 10p 13p 1 4p 5p 5p 8s 4sh 5p 5s 4sh Parsely 20 9p 4s 2p 9p 11p 8p 0 4p 5p 4p 6s 5sh 5p 2s 2sh Cabbage 20 0 2p 3sh 0 1p 3p 2p 2p 2p 0 0 2sh 2p 2p 2sh Tomatoes 20 0 1p 2sh 0 3p 3p 0 0 0 2p 2p 2p 0 4p 0 Green onions 20 1p 2p 4sh 0 4p 4p 0 0 4p 0 0 4p 0 0 0 p: Proteus spp., s: Salmonella spp., sh: Shigella spp. BS: BeyşehirMarket Place, DH: Doğanhisar Market Place, IL: IlgınMarket Place Table III. Survival periods (days) of microorganisms on the vegetable specimens collected from Beysehir, Doğanhisar and Ilgınmarkets in Konya Province, (n=5) Vegetable varieties Microorganisms Green onion Tomatoes Cabbage Parsely Watercress Radish Carrots Cucumber (skin) Lettuce Green pepper Mean Shigella spp 3 2.5 4 3 5 2 3 2.5 6 2 3.3 Salmonella spp 10.5 4 3 16 14 3.5 6 5 15 5 8.2 Proteus spp 16 14 11 17.5 16 11 9 13 19 8.5 13.45 Escherichia coli 10.5 4 6 9.5 10 5 3 3 10 2 6.3 Coliform 18 15 14 20 20 13 12 15 19.5 10 15.55 Mean 11.6 7.9 7.6 10.4 12.9 6.9 6.6 7.7 13.7 5.5  When the market places were compared to each other, in terms of the distribution of the microbiological pollution, the highest incidence of coliform contamination (39-50 %) with the IlgınMarket and it was followed by the Doğanhisar (37-50 %) and Beysehir (36-50 %) market places, respectively (Table I). These findings were coincided with the observations stated above that the hygiene precautions taken were found to be very deficient in the IlgınMarket and the sanitary conditions in the Doğanhisar Market were in sufficient. Whereas, the Main Market Place of Konya city has been supposed to have higher levels of hygiene then the other two markets.  As far as the pathogenic bacterial pollution was concerned, only three pathogenic genera were isolated from the vegetable specimens studied, and they were Proteus sp., Salmonella sp. and Shigella sp. The highest incidence of pathogenic pollution was found to be related to the genus of Proteus and it was followed by the genera of Salmonella and Shigella spp. respectively. On the other hand, the microbiological pollution of the pathogenic microorganisms given above showed nearly similar distribution patterns among both the vegetable specimens and the market places studied to those in the case of coliform contamination as shown in the Table I and II.  The results stated above could indicate high percentages of coliforms but low percentages of pathogenic microorganisms and thus fecal pollution of vegetables which are eaten fresh.  It was found that the bacterial coliforms most frequently encountered were Esherichia coli and Enterobacter aerogene. Whereas, with respect to potential pathogens, microorganisms of the genera of Proteus, Salmonella and Shigella were detected.  When the treatment methods tested, were compared to each other, it was found that the “scalding” was the most effective method to disinfect the vegetable samples studied among the four treatment methods tested and then it was followed by the treatment methods of “washing with a bacteriocide” and “washing with a detergent” respectively. On the other hand, the treatment method of “washing with water” was found to have not any significant effect upon the disinfection of the vegetable samples studied comparing to the treatment method of “No Treatment”, in the experiment.  The longest survival period on the vegetable samples examined, was observed with the bacteria of coliform (mean: 15 days) and then it was followed by Proteus spp (13 days), Salmonella spp (8 days), Esherichia coli (8 days) and Shigella spp (3 days) respectively. On the other hand, it was found that the microorganisms had a general tendency to have the longest survival time with the samples of lettuce plant (mean: 13 days) and then it was followed by the watercress (13 days), green onion (11 days), parsley (10 days), tomatoes (8 days), cucumber skin and cabbage (7 days), radish (7 days), carrots (7 days) and green pepper (5 days) vegetable samples, respectively (Table III). These findings were supported by the results of Larkin et. al. [12] and Rudolfs et. al. [13] who have carried out several field experiments with vegetables irrigated with municipal wastewaters and thus, suggested that the leafy vegetables had a general tendency to have larger spaces to host higher number of microorganisms on their surfaces.  The results obtained from this study were generally supported by various works given in the literature.

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